CCR, LGI, Flow Cytometry Core

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

The mission of the CCR LGI Flow Cytometry Core in Building 37 (FC37) is to offer up-to-date instrumentation and technical expertise to CCR investigators to assist cancer research. The core provides full-scale, state-of-the-art flow cytometry services including analytical sample acquisition, data analysis, imaging flow cytometry, fluorescent cell sorting and experimental planning and consultation. The core is committed to the dissemination of novel flow cytometry-based technologies through continuous education of investigators and participating in the NCI-wide flow cytometry training course with active contributions from director Ferenc Livak and staff members, Karen W. Wolcott and Caiyi Li. These efforts were recognized in December 2020 with the NCI Director's Merit Award which was presented to participating members of the NCI flow cytometry cores and facilities. The Core also contributed to the proposal of hiring a shared bioinformatics expert to support high dimensional flow cytometry data analysis at NCI. This full time contract position has been approved and the search began for a suitable candidate. The LGI FC37 provides instrumentation for a wide variety of flow cytometry technologies. The core is equipped with six analytical instruments: a state-of-the-art BD FACSymphony A5 flow cytometer equipped with five lasers and the capacity to identify 27 fluorochromes also equipped with a high throughput sampler (HTS) attachment that accommodates 96-well plates to further increase the capacity of the instrument. Two high end BD LSRFortessa cytometers with identical configuration, equipped with five lasers and the capacity to identify 18 fluorochromes. One of these instruments, LSRFortessa SORP1 is also equipped with HTS attachment. The core also has one three-laser, digital BD FACSCanto II cytometer and one, 32-channel Sony SA3800 spectral analyzer. In addition, the Core operates a dual-camera, 4-laser ImageStream MarkII imaging flow cytometer equipped with HTS attachment. To support cell sorting the Core operates two BD FACSAria II instruments, equipped with 3-4 lasers to simultaneously identify 11-12 fluorochromes. Both of these sorters are enclosed in flexible, BioBubble Benchtop Biocontainment units. One Beckman Coulter MoFlo Astrios EQ instrument, housed in a custom-designed Class II biosafety cabinet, is equipped with five lasers and capable of detecting 19 fluorochromes. One BD FACSAria Fusion cell sorter is also housed in a custom-designed Class II type A2 biosafety cabinet and is equipped with five lasers and has the capacity to identify 18 fluorochromes. The two Class II biosafety cabinets and two BioBubble Benchtop Biocontainment units, combined with the separate housing in a dedicated laboratory space in Room 6008A allow the LGI FC37 to operate in full compliance with NIH Policy for Biosafety of Cell Sorters (July 28, 2012), to perform all sorts at a BSL-2 with enhanced precautions aerosol containment level. In FY21 the Core has undergone three major changes. Director Ferenc Livak wrote a successful RRS application for a new spectral analyzer which was approved. This Sony ID7000 spectral analyzer has been purchased and will replace the older Sony SA3800 analyzer. The ID7000 represents the most advanced flow cytometry analyzer currently available in the World with 5 lasers and a 144 channel detector array and will provide significant help in support of high dimensional flow cytometry analyses at NCI. The Core also decided to replace its aging, expensive and underused MoFlo Astrios cell sorter with a new Sony MA900 cell sorter. This sorter was acquired by CCR SCAF last year with support from the Core, but remained poorly utilized and is now being transferred to the LGI FC37 main laboratory. This simpler and highly automated cell sorter will effectively increase sorting capacity at a time when staff reduction due to the pandemic has caused major restrictions on cell sorter scheduling. Also in response to the space constrains which were further exacerbated by the COVID-19 pandemic, CCR agreed to significant space expansion for the Core. Director Ferenc Livak and staff member Karen W. Wolcott have worked with James Galantine and his team at NCI Office of Space and Facilities Management to develop a project to accommodate all the needs and safety requirements of the extended facility. This expansion is scheduled to take place in FY22. Much consideration has been given to ensure that instrument users can maintain the new norm of 6-foot physical distancing. The expanded space will also give the Core the opportunity to introduce another, high-end cell sorter in the coming years in a separate lab space that can continue to operate even in case of future emergency situations. Despite the significant disruptions caused by the COVID-19 pandemic, the LGI FC37 served 238 users from 82 CCR laboratories as well as from 4 additional laboratories of four other NIH institutes in FY21. Several of these laboratories heavily depend on the services of the Core in conducting high-dimensional analyses aimed at better understanding of lymphocyte development (Ashwell, Bosselut (LICB] and Bhandoola [LGI] labs) and tumor-associated immune responses (Goldszmid lab [CIP] and Zhuang lab [NOB]). Single cell sorting helps the rapid generation of CRISPR-induced mutant cell lines and is becoming an essential tool in single cell genomic analyses and CRSIPR library screening (Casellas lab, [NIAMS/NCI]). The LGI FC37 provides critical help to the initial phases of translational research into lymphoma targeting screens (Staudt lab [LMBr]), glioblastoma research (Gilbert, Chunzhang Yang labs [NOB]) and tumor-specific immune responses (Li Yang, Merlino labs [LCBG] and Waldman lab [LMBr]. The LGI FC37 has trained 72 new users in FY21 on analytical instruments and has also trained one new user for cell sorting. New users are instructed to review instrument and software training tutorials before attending a 4-hor hands-on training session that includes startup and shut down procedures for the cytometers, setting up experiments, basic instrument troubleshooting, and data analysis. Importantly, staff member Karen Wolcott has successfully adapted a Webex-based remote training schedule that allows the Core to offer training while observing the NIH guidelines on physical distancing. Core staff also provides follow-up assistance and helps new users develop confidence in using the technology correctly. Cell sorter training is offered to select users who need frequent, possibly after hour sorting time. The LGI FC37 employs an entirely online scheduling and record keeping platform using the NIH LabShare application. Appropriate policies are in place to ensure safe, fair and equitable access of Core resources to all registered users. After June 22nd 2020, during the first phase of reopening of NIH, the Core began operating at approximately 50% capacity, with two staff members on site for three days a week and offering a 6-day/week support. Analyzer and cell sorter support was reduced to the extent that was consistent with the NIH social distancing guidelines. By now every Core staff member has been vaccinated and this allowed the Core to restore nearly full staff operations (4 out of 5 members) from June 1st 2021, to alleviate the significant pressure on cell sorter scheduling. Analyzer use remains limited to the extent that is consistent with the NIH social distancing guidelines currently in effect on the Bethesda campus. Despite these challenges, the LGI FC37 remains committed to be a leader of flow cytometry services at CCR by offering the highest quality, reliable support to the largest number of NCI investigators on campus The LGI FC37 is dedicated to the introduction of innovative flow cytometry-based technologies to further advance the cutting-edge cancer research conducted at NIH.